Synthesis and Evaluation of Structurally Constrained Quinazolinone

J. Med. Chem. 2008, 51, 6889–6901

6889

Synthesis and Evaluation of Structurally Constrained Quinazolinone Derivatives as Potent and Selective Histamine H3 Receptor Inverse Agonists Tsuyoshi Nagase, Takashi Mizutani, Etsuko Sekino, Shiho Ishikawa, Sayaka Ito, Yuko Mitobe, Yasuhisa Miyamoto, Ryo Yoshimoto, Takeshi Tanaka, Akane Ishihara, Norihiro Takenaga, Shigeru Tokita, and Nagaaki Sato* Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba 300-2611, Japan ReceiVed May 15, 2008

A series of structurally constrained derivatives of the potent H3 inverse agonist 1 was designed, synthesized, and evaluated as histamine H3 receptor inverse agonists. As a result, the N-cyclobutylpiperidin-4-yloxy group as in 2f was identified as an optimal surrogate structure for the flexible 1-pyrrolidinopropoxy group of 1. Subsequent optimization of the quinazolinone core of 2f revealed that substitution at the 5-position of the quinazolinone ring influences potency. Representative derivatives 5a and 5s showed improved potency in a histamine release assay in rats and a receptor occupancy assay in mice. Introduction Histamine plays a variety of physiological roles in the CNSa and peripheral tissues. In the CNS, histaminergic neurons are exclusively localized in the tuberomammillary nucleus of the hypothalamus but project widely throughout the CNS.1 There are four known G protein-coupled receptors for histamine: H1, H2, H3, and H4.2 The histamine receptors have unique signal transduction pathways and distribution, which has led to the discovery of a variety of physiological roles for histamine. Of these receptors, H3 is predominantly expressed in the CNS, while H1 and H2 are expressed in both central and peripheral tissues.3,4 The H4 receptor is predominantly expressed in inflammatory cells, suggesting its critical role in the regulation of inflammatory and immune responses.5 The H3 receptor was discovered pharmacologically in 19836 and genetically identified in 1999.3 The genetic identification of the H3 receptor gained much attention and redirected both the detailed pharmacological characterization of the receptor and efforts from academia to the pharmaceutical industry to find drugs that bind specifically to H3.7 Signaling through the H3 receptor activates G proteins that inhibit adenylate cyclase activity and reduce intracellular cAMP levels.3,8 In the CNS, the H3 receptor is localized on the presynaptic membrane as an autoreceptor and negatively regulates the release and synthesis of histamine.6 In addition, the H3 receptor is known to modulate the release of other neurotransmitters such as norepinephrine, dopamine, acetylcholine, serotonin, and GABA.9 The H3 receptor signals constitutively, which serves to tonically suppress target neuronal activities such as histamine release to baseline levels.10 Agonist-induced signaling that occurs in the presence of elevated histamine levels further suppresses histamine release. While classical antagonists would interfere with histamine-mediated negative feedback, H3 receptor inverse agonists have been demonstrated to decrease constitutive H3 signaling, thus blocking the tonic inhibition of histamine release and further potentiating histaminergic effects. Because * To whom correspondence should be addressed. Phone: +81-29-8772004. Fax: +81-29-877-2029. E-mail: [email protected]. a Abbreviations: P-gp, P-glycoprotein; SD rat, Sprague-Dawley rat; SAR, structure-activity relationship; hERG, human ether-a-go-go-related gene; HEK, human embryonic kidney; CNS, central nervous system; Ts, 4-toluenesulfonyl; Ms, methanesulfonyl; DIAD, diisopropyl azodicarboxylate; DEAD, diethyl azodicarboxylate.

Figure 1. Structures and antagonistic activities of nonimidazole H3 antagonists and inverse agonists. Antagonistic activities were determined by inhibition of R-R-methylhistamine-induced binding of [35S]GTPγS at human H3 receptor. The values represent the mean ( SE for n g 3.

of the effects of H3 signaling on multiple neuronal transmitters, it has been suggested that H3 antagonists/inverse agonists could be effective therapeutics for several CNS-related disorders.11 In animal models, H3 receptor antagonists/inverse agonists have been shown to enhance wakefulness and attentive and cognitive behavior while reducing feeding and body weight.12,13 Moreover, very recently it has been reported that 25 (BF2.649) (Figure 1), a potent and selective H3 receptor inverse agonist, suppressed the excessive daytime sleep of narcoleptic patients.14 First-generation imidazole-based H3 antagonists have inhibitory activity on cytochrome P450 enzymes, which may result in drug-drug interactions with coadministered drugs by reducing hepatic clearance.15 Current efforts have focused on nonimidazole classes of H3 receptor antagonists/inverse agonists with the potential to reduce these liabilities. Since the identification of the H3 receptor genes, various classes of nonimidazole H3 receptor antagonists have been developed to target H3 receptors in the CNS.7,12,16 Several of them, including 25,14,17 26 (ABT-239),18 and 27 (GSK189254)19 (Figure 1), have entered clinical trials to evaluate their effectiveness in treating CNS disorders such as excessive daytime sleepiness, schizophrenia, and cognitive dysfunction. Starting from the previously reported potent H3 inverse agonist 1 (Figure 1),20 a series of structurally constrained analogues were prepared and evaluated, aimed at improving its potency. This approach was previously demonstrated by the

10.1021/jm800569w CCC: $40.75  2008 American Chemical Society Published on Web 10/08/2008

6890 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21

Nagase et al.

Scheme 1a

a

Reagents: (a) DEAD, PPh3, THF; (b) trifluoroacetic acid, CHCl3; (c) R2R3CHI, K2CO3, DMF, 60 °C; (d) R2R3CO, ZnCl2, NaBH3CN, MeOH.

Scheme 2a

a Reagents: (a) 6, DIAD, PPh3, THF; (b) MsCl, Et3N, CH2Cl2; (c) pyrrolidine, K2CO3, DMF, 80 °C; (d) DIAD, PPh3, PhCO2H, THF; (e) K2CO3, MeOH.

Scheme 3a

a

Reagents: (a) 6, DIAD, PPh3, THF; (b) 10% HCl aq, THF; (c) pyrrolidine, ZnCl2, NaBH3CN, MeOH.

Johnson & Johnson group, in which an N-alkylpiperidin-4-yloxy group was systematically investigated as a structurally constrained replacement for the amimopropoxy group and an N-isopropylpiperidin-4-yloxy group was successfully identified.21 In the present SAR study, additional novel structurally constrained tethers were designed and synthesized to replace the flexible aminopropoxy group of 1, and the N-cyclobutylpiperidin-4-yloxy group22 as in 2f was identified as a useful surrogate structure. The quinazolinone core of 2f was subsequently optimized to find potent analogues 5a-s. Compounds 5a and 5s were profiled in vivo and shown to have good pharmacokinetic profiles and potent efficacy. In this report, the design, synthesis, SAR of the structurally constrained quinazoli-

none class of H3 inverse agonists, and in vivo evaluation of the potent derivatives 5 are described. Chemistry The synthetic route for the derivatives reported herein is described in Schemes 1-4. Scheme 1 outlines the synthesis of compounds 9b and 2a-l. Known compounds 623 and 7 were coupled by the Mitsunobu reaction24 to give ethers 8, followed by deprotection of the tert-butoxycarbonyl group to furnish amines 9. The amino group of compounds 9 was alkylated with the desired iodoalkane (method c) or reductively alkylated with the desired ketones in the presence of sodium cyanoborohydride and zinc chloride (method d) to furnish 2a-l. Preparation of

Quinazolinones as Histamine H3 Receptor InVerse Agonists

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6891

Scheme 4a

a Reagents: (a) 7b, NaH, DMF; (b) trifluoroacetic acid, CHCl3; (c) cyclobutanone, ZnCl2, NaBH3CN, MeOH; (d) (i) H2, 10% Pd/C, MeOH, (ii) TsOH · H2O; (e) AcOH, rt.

analogue 3a and its corresponding diastereomer 3b is illustrated in Scheme 2. Cyclopentane-1,3-diol (10) and compound 6 were coupled to give alcohol 11 as a single diastereomer in 99% yield. Mesylation of the hydroxy group of 11 was followed by the displacement with pyrrolidine to furnish amine 3a in good yield. The corresponding diastereomer 3b was prepared by the inversion of the hydroxy group configuration in compound 11 by the Mitsunobu protocol. The benzoate group of product 13 was cleaved, and the resulting hydroxy group was mesylated and displaced with pyrrolidine to give diastereomer 3b in good yield. The relative stereochemistry of diastereomers 3a and 3b has not yet been established. The preparation of compound 4 is shown in Scheme 3. Coupling reaction between 4,4-ethylenedioxycyclohexanol (16) and 6, followed by hydrolysis of the ketal under acidic conditions, afforded the ketone 18, which was reductively aminated with pyrrolidine to give amines 4a and 4b after the separation of the diastereomeric mixture. The quinazolinone ring-modified derivatives were conveniently prepared as depicted in Scheme 4. Compound 7b was coupled with 4-fluoronitrobenzene (19) in the presence of sodium hydride followed by deprotection of the tert-butoxycarbonyl group to afford the amine 21. Reductive alkylation of piperidine 21 with cyclobutanone followed by reduction of the nitro group of intermediate 22 gave the aniline 23, which was conveniently isolated as a tosylate salt. Finally, the amino group of compound 23 was reacted with the desired substituted- or aza-benzoxazinones 24a-s20,25 to furnish the derivatives 5a-s. Results and Discussion The compounds were tested using the [35S]GTPγS binding assay in membranes isolated from cells transfected with cloned H3 receptors.20 All the quinazolinone derivatives reported herein reduced the basal GTPγS binding, indicating that they are inverse agonists. Selected compounds were evaluated for hERG K+ channel inhibitory activity in the [35S]N-[(4R)-1′-[(2R)-6cyano-1,2,3,4-tetrahydro-2-naphthalenyl]-3,4-dihydro-4hydroxyspiro[2H-1-benzopyran-2,4′-piperidin]-6-yl]methanesulfonamide binding assay to assess cardiac QTc prolongation liability.26 We recently discovered the quinazolinone class of histamine H3 receptor inverse agonists and identified the clinical development candidate 1 (Figure 1).20 The essential pharmacophore of the quinazolinone derivatives is the aminopropoxyphenyl portion that is a common structural motif recognized by a number of research groups. We envisioned that the potency

of compound 1 (hH3, IC50 ) 1.7 nM) (Figure 1) might be further improved by imparting structural constraints to the flexible propoxy moiety. In addition to the previously studied N-alkylpiperidin-4-yloxy group,21 novel structurally constrained tethers were designed to have a cyclic structure in the aminopropoxy region, conserving the three-carbon tether between the oxygen and nitrogen atoms of the aminopropoxy moiety (Table 1). First, we prepared a variety of N-substituted piperidinyloxy derivatives 2a-h. N-Substitution is essential for activity because the nonsubstituted derivative 9b is devoid of potency. The ethyl derivative 2b displayed the highest activity (IC50 ) 49 nM) among the unbranched alkyl derivatives 2a-d. Further extension of the alkyl chain was not attempted due to the decreased binding affinities of analogues 2c and 2d. The isopropyl derivative 2e was 3-fold more potent than the ethyl derivative 2b. The cyclobutyl derivative 2f (IC50 ) 1.5 nM) showed a 10-fold improvement over 2e, while ring expansion as in analogues 2g and 2h resulted in decreases in potency. Second, the piperidine was replaced by pyrrolidine and azacycloheptane (2i-l), which affects the direction of the N-alkyl substituent. The cyclobutyl and cyclopentylpyrrolidine derivatives (2i and 2j) displayed significantly decreased activities. The azacycloheptane derivatives 2k and 2l were somewhat less potent than the corresponding piperidine derivatives 2f and 2g. Further ring expansion was not attempted because undesirable hERG activity was observed for compounds 2k and 2l, possibly attributed to an increase in lipophilicity for those analogues. Finally, the nitrogen atom was incorporated in the distal ring as in cycloalkyl analogues 3 and 4. The cyclopentyl derivative 3a showed a potent H3 activity (IC50 ) 3.4 nM) with a satisfactory hERG activity. The corresponding diastereomer 3b was less potent. The cyclohexyl derivatives 4a and 4b were prepared even though they have a four-carbon tether. The transisomer 4a showed good activity despite its four-carbon tether, and the corresponding cis-isomer 4b was significantly less potent than 4a. In this structurally constrained design, the cyclobutylpiperidinyloxy structure 2f was identified as a potent substructure. It should be noted that racemic 3a has the potential for additional improvement by modification of the size of the two rings. Further optimization of 2f by the modification of the quinazolinone group is summarized in Table 2. The fluorinesubstituted derivatives 5a-e displayed improved activities

6892 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 Table 1. In Vitro Profiles of Compounds 1, 2-4, and 9ba

Nagase et al. Table 2. In Vitro Potency of Compounds 5a-sa

compd

R

human H3b (IC50, nM)

hERG (IC50, µM)c

2f 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s

H 5-F 6-F 7-F 8-F 6,7-diF 5-Cl 6-Cl 7-Cl 8-Cl 5-OCH3 6-OCH3 7-OCH3 8-OCH3 5-Aza 6-Aza 7-Aza 8-Aza 5-CH3 5-CF3

1.5 ( 0.3 0.55 ( 0.04 1.2 ( 0.1 1.3 ( 0.1 0.84 ( 0.12 0.91 ( 0.06 0.34 ( 0.14 1.5 ( 0.2 3.0 ( 0.5 1.7 ( 0.1 1.2 ( 0.3 1.5 ( 0.1 1.8 ( 0.4 4.2 ( 1.0 14 ( 1 2.0 ( 0.4 1.8 ( 0.2 1.6 ( 0.2 0.27 ( 0.08 0.33 ( 0.05

>10 >10 >10 >10 >10 >10 >10 5.4 ( 0.2 3.9 ( 0.3 3.3 ( 0.8 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

a The values represent the mean ( SE for n g 3. b Inhibition of R-Rmethylhistamine-induced binding of [35S]GTPγS at human H3 receptor. c Inhibition of [35S]N-[(4R)-1′-[(2R)-6-cyano-1,2,3,4-tetrahydro-2-naphthalenyl]-3,4-dihydro-4-hydroxyspiro[2H-1-benzopyran-2,4′-piperidin]-6-yl]methanesulfonamide binding to hERG in HEK293 cells.

Table 3. Pharmacokinetic Parameters of 5a, 5r, and 5sa compd

species

IV Clp (mL/min/kg)

PO AUC0-∞ (µM · h)

Cmax (µM)

F (%)

5a

rat dog monkey rat dog monkey rat dog monkey

11 44 27 25 17 17 24 13 26

6.2 0.05 1.7 2.33 5.6 0.08 1.66 1.3 0.15

1.3 0.02 0.38 0.56 1.1 0.02 0.42 0.28 0.05

53 4.8 37 40 76 1 33 46 11

5r

5s

a The reported data are an average generated after 1 mg/kg iv and 3 mg/kg po doses in n ) 3 animals/dose for rats and 0.3 mg/kg iv and 1 mg/kg po doses in n ) 3 animals/dose for dogs and monkeys.

a The values represent the mean ( SE for n g 3. b Inhibition of R-Rmethylhistamine-induced binding of [35S]GTPγS at human H3 receptor. c Inhibition of [35S]N-[(4R)-1′-[(2R)-6-cyano-1,2,3,4-tetrahydro-2-naphthalenyl]-3,4-dihydro-4-hydroxyspiro[2H-1-benzopyran-2,4′-piperidin]-6-yl]methanesulfonamide binding to hERG in HEK293 cells. d Not determined.

compared to the parent 2f. The 5-fluoro 5a (IC50 ) 0.55 nM) was the most potent derivative. In the chloro derivatives 5f-i, the 5-chloro derivative 5f was 4-fold more potent than the parent 2f, while chloro substitution at the other positions (5g-i) was

not effective in improving potency. The derivatives with an electron-donating methoxy substituent (5j-m) did not show noticeable improvement. Regarding the aza derivatives, the 5-aza derivative 5n showed a significant decrease in potency while the other aza derivatives 5o-q were tolerated. Encouraged by the improved potency demonstrated by the 5-chloro and fluoro derivatives (5a and 5f), 5-methyl and 5-trifluoromethyl derivatives 5r and 5s were prepared and found to possess 5-fold more potent activities than 2f. This enhancement of potency by the 5-substitution of the quinazolinone ring is specific to these N-cyclobutylpiperidinyloxy derivatives and was not observed in the flexible propoxy derivatives.20 Compounds 5a, 5r, and 5s were demonstrated to reduce basal GTPγS binding with EC50 values of 1.1, 0.34, and 0.71 nM, respectively, indicating that these three compounds are inverse agonists. The representative potent 5-substituted derivatives 5a, 5r, and 5s were evaluated in pharmacokinetic studies (Table 3). All three compounds showed suitable pharmacokinetic profiles for in vivo evaluation in SD rats. The systemic exposure of compound 5a in rats was much higher (AUC0-∞ ) 6.2 µM · h, Cmax ) 1.3 µM) than those of compounds 5r and 5s. The pharmacokinetic profile of compound 5a in dogs, however, was very poor, which



Table 4. Brain Penetration and P-gp Susceptibility of 5a, 5r, and 5s brain penetration in SD ratsa

P-gp susceptibilityb ratio

transcellular transport ratio (B-to-A)/(A-to-B)

compd

plasma (µM)

brain (nmol/g)

CSF (µM)

brain/plasma

CSF/brain

mouse mdr1a

human MDR1

5a 5r 5s

2.4 1.4 0.7

7.6 3.2 2.3

0.95 0.10 0.11

3.2 2.3 3.3

0.13 0.03 0.05

1.8 1.0 2.5

1.0 0.9 0.7

a The concentrations were determined at 2 h after 10 mg/kg oral administration. The values represent the mean for n ) 3 animals. b Transcellular transport ratio ((B-to-A)/(A-to-B)) in human MDR1- and mouse mdr1a-transfected LLC-PK1 cell line. The values represent the mean for n ) 3. The value above 3.0 indicates that a compound is a P-gp substrate.

Table 5. Binding Affinities of Compounds 5a, 5r, and 5s for the Human, Rat, and Mouse H3 Receptorsa H3 binding affinity (Ki, nM)b compd

human

rat

mouse

5a 5r 5s

6.8 ( 1.9 1.7 ( 0.4 3.6 ( 0.7

6.0 ( 0.7 1.8 ( 0.1 2.3 ( 0.1

8.4 ( 1.0 3.9 ( 0.6 3.9 ( 0.5

a The values represent the mean ( SE for n g 3. b Displacement of [3H]N-R-methylhistamine binding to cell membranes expressing recombinant H3 receptors.

is most likely due to the significantly higher clearance value. Compounds 5r and 5s showed good systemic exposure and oral bioavailability in dogs. In contrast, the pharmacokinetic profiles of 5s and 5r in monkeys were poor, whereas 5a exhibited a fair profile in monkeys. It should be noted that the clearance value of 5a in monkeys is the highest (Clp ) 27 mL/min/kg) among the three derivatives, so the poor pharmacokinetic profiles of 5r and 5s may be caused by monkey-specific absorption problems. The compounds were studied for their ability to penetrate the CNS using several assays. The brain and cerebrospinal fluid (CSF) levels in SD rats 2 h following 10 mg/kg oral administration of 5a, 5r, and 5s were examined (Table 4). All the derivatives displayed good brain and CSF exposure. P-gp susceptibility was evaluated28 as a pivotal factor for brain penetration, and none of them were found to be substrates for human P-gp (MDR1) (Table 4). Compounds 5a and 5s might be very weak substrates for mouse P-gp (mdr1a); see further discussion below regarding mouse P-gp susceptibility. Compounds 5a and 5s were confirmed to have excellent selectivity with respect to other histamine receptor subtypes (IC50 > 10 µM for hH1, hH2, and hH4) and to a panel of 115 diverse, unrelated binding sites (IC50 >1 µM for all the binding sites tested). The binding affinities of compounds 5a, 5r, and 5r for the human, rat, and mouse H3 receptors are summarized in Table 5. The compounds showed potent binding affinities, and no significant species difference in binding affinity was observed. Having demonstrated excellent potency, selectivity, pharmacokinetic profile, and brain penetration, 5a and 5s were tested for brain histamine release in SD rats. In our histamine release assay,29 the test compound (po) and pargyline (ip), a monoamine oxidase inhibitor, were codosed in SD rats. After 2 h, the whole brain was rapidly removed, and the concentration of telemethylhistamine, a major extracellular metabolite of histamine, was measured. Significant and dose-proportional elevation of tele-methylhistamine was observed in the rat brain after oral administration of 5a and 5s (Figure 2). Brain histamine H3 receptor occupancy was evaluated by an ex vivo receptor occupancy method for correlation with the observed histamine elevation. The brain H3 receptor was shown to be highly occupied (>90%) 2 h following oral administration of 1 mg/kg of 5a and 5s. A high degree of receptor occupancy

seems to be necessary for the H3 inverse agonists to exhibit significant elevation of brain histamine in rats.30 As a first step in estimating efficacious plasma levels in humans, we decided to study receptor occupancy of 5a and 5s in mice in detail. As shown in Table 4, 5a and 5s are not human P-gp substrates but may be weak mouse P-gp substrates. Hence, brain exposure and receptor occupancy studies in P-gp-deficient mdr1a (-/-) and wild type mdr1a (+/+) CF-1 mice31 were carried out. The plasma and brain concentrations were determined 2 h after oral administration of 0.3 mg/kg of 5a and 1 mg/kg of 5s in mdr1a (-/-) and mdr1a (+/+) CF-1 mice (Table 6). The comparison of the brain-to-plasma ratios in mdr1a (-/-) and mdr1a (+/+) CF-1 mice clearly suggests that P-gpmediated efflux has a considerable influence on brain penetration of 5a and 5s in CF-1 mice. The brain-to-plasma ratios for 5a and 5s in mdr1a (-/-) CF-1 mice were 9.4 and 13, which are about 5-fold higher than those in mdr1a (+/+) CF-1 mice (1.9 for 5a and 2.6 for 5s). Next, the receptor occupancy-brain concentration relationships were studied in CF-1 mice (Figure 3). The brain concentrations that are required to achieve 90% receptor occupancy (brain Occ90) for 5a and 5s were determined to be 0.28 and 0.15 nmol/g, respectively. The plasma Occ90 values were estimated from the brain Occ90 values and the brain-toplasma ratios (plasma Occ90 ) brain Occ90/brain-to-plasma ratio), and the results are shown in Table 6. The estimated plasma Occ90 values in Table 6 suggest that the plasma Occ90 values in humans might be as low as 30 nM for 5a and 11 nM for 5s. On the basis of the human P-gp transcellular transport ratios for 5a and 5s, it is assumed that the effect of P-gpmediated efflux is negligible in humans. Investigation of receptor occupancy in other higher species by noninvasive receptor occupancy determination methods such as the positron emission tomography is necessary to confirm these predictions. Examination of the relationship between the degree of receptor occupancy and efficacy in animal models using 5a and 5s is in progress. Summary A series of structurally constrained analogues of potent H3 inverse agonist 1 were designed, synthesized, and evaluated as histamine H3 receptor inverse agonists. As a result, the N-cyclobutylpiperidin-4-yloxy group as in compound 2f was identified as an optimal surrogate structure for the flexible 3-(1pyrrolidinyl)propoxy group of lead compound 1. Subsequent optimization of the quinazolinone 2f revealed that substitution at the 5-position of the quinazolinone ring markedly enhances in vitro potency. Representative analogues 5a and 5s had excellent selectivity with respect to other histamine receptor subtypes and to 115 diverse unrelated binding sites. Compounds 5a and 5s showed satisfactory pharmacokinetic profiles and brain penetrability in the preclinical animals. Two hours following oral administration of 5a and 5s in SD rats, a dose-proportional and statistically significant increase of brain histamine levels


Nagase et al.

Figure 2. Brain tele-methylhistamine levels in SD rats after oral administration of 5a and 5s. Values are means ( SE, determined from five experiments. * P < 0.05 and ** P < 0.01 (ANOVA Dunnett) compared with the vehicle control. Table 6. Brain Penetration and Plasma Occ90 Values of 5a and 5s in P-gp-deficient mdr1a (-/-) and Wild Type mdr1a (+/+) CF-1 Micea compd 5a 5s

mdr1a (+/+) mdr1a (-/-) mdr1a (+/+) mdr1a (-/-)

plasmab (µM)

brainb (nmol/g)

0.032 ( 0.002 0.028 ( 0.001 0.012 ( 0.001 0.010 ( 0.002

0.062 ( 0.002 0.260 ( 0.039 0.031 ( 0.002 0.137 ( 0.014

brain/ plasma plasmac Occ90d (nM) 1.9 9.4 2.6 13

144 30 58 11

a The brain and plasma concentrations were obtained 2 h following oral administration of 5a (0.3 mg/kg) and 5s (1 mg/kg) in mice. b The values represent the mean ( SE for n ) 3. c The ratios were obtained from the mean values. d Plasma Occ90 was calculated by brain Occ90/brain to plasma ratio. The brain Occ90 values for 5a and 5s are 0.28 nmol/g and 0.15 nmol/ g, respectively (Figure 3).

Figure 3. The brain concentration-receptor occupancy relationship of (a) 5a and (b) 5s in CF-1 mice. Receptor occupancy and exposure were determined 2 h following oral administration of vehicle or compound 5a or 5s (0.1, 0.3, 1, and 3 mg/kg). See Supporting Information for experimental details.

was observed, indicating that the brain H3 receptors were highly occupied (>90%). The plasma Occ90 for 5s in humans was estimated to be as low as 11 nM based on P-gp susceptibility and the receptor occupancy studies in P-gp-deficient mdr1a (-/-) and wild type mdr1a (+/+) CF-1 mice. The potential cardiovascular effects of compound 5s were evaluated in anesthetized and ventilated dogs.27 At 3 mg/kg iv dosing (Cmax ) 3.2 µM), no adverse treatment-related cardiovascular effects were observed. Regarding gross behavior in mice, oral admin-

istration of compound 5s at a dose of 100 mg/kg caused no treatment-related changes in psychomotor activities, motor activities, muscle tone, CNS excitation, autonomic responses, and reflexes.27 Further profiling of 5a and 5s is in progress to select an improved clinical candidate from this class, and updated results will be reported in due course. Experimental Section Chemistry. General Procedures. Unless otherwise noted, all solvents, chemicals, and reagents were obtained commercially and used without purification. The 1H NMR spectra were obtained at 400 MHz on a MERCURY-400 (Varian) or JMNAL400 (JEOL) spectrometer, with chemical shift (δ, ppm) reported relative to TMS as an internal standard. Mass spectra were recorded with electron-spray ionization (ESI) or atmospheric pressure chemical ionization (APCI) on a Waters micromass ZQ, micromass Quattro II, or micromass Q-Tof-2 instrument. Flash chromatography was carried out with prepacked silica gel columns (KP-Sil silica) from Biotage. Preparative thin-layer chromatography was performed on a TLC Silica gel 60 F (Merck KGaA). Preparative HPLC purification was carried out on a YMC-Pack Pro C18 (YMC, 50 mm × 30 mm id), eluting with a gradient of CH3CN/0.1% aqueous CF3CO2H at a flow rate of 40 mL/min. Purity of target compounds was determined by HPLC with the two different eluting methods as follows. Analytical HPLC was performed on a SPELCO Ascentis Express (4.6 mm × 150 mm id), eluting with a gradient of (A) 0.1% H3PO4/CH3CN ) 95/5 to 10/90 over 7 min followed by 10/90 isocratic over 1 min and (B) 10 mM potassium phosphate buffer (pH 6.6)/CH3CN ) 95/5 to 20/80 over 7 min followed by 20/80 isocratic over 1 min (detection at 210 nm). High resolution mass spectra were recorded with electron-spray ionization on a micromass Q-Tof-2 instrument. General Procedure for the Preparation of 2a–e via Alkylation. 3-(4-[(1-Ethyl-4-piperidinyl)oxy]phenyl)-2-methyl-4(3H)-quinazolinone (2b). To a mixture of 9b (50 mg, 0.15 mmol) and potassium carbonate (42 mg, 0.30 mmol) in dry DMF (1 mL) was added ethyl iodide (23 mg, 0.15 mmol) under a nitrogen atmosphere, and the mixture was stirred at room temperature for 1 h. The resulting mixture was partitioned between ethyl acetate and 1 N NaOH. The layers were separated, and the aqueous layer was extracted with ethyl acetate twice. The combined organic layers were washed with 1 N NaOH and brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography with 3% CHCl3 in methanol to give 2b as a colorless solid (25 mg, 46%); HPLC purity (99.2%). 1H NMR (400 MHz, CDCl3) δ 1.12 (3H, J ) 6.8 Hz), 1.83-1.95 (2H, m), 2.02-2.10 (2H, m), 2.26 (3H, s), 2.26-2.39 (2H, m), 2.45 (2H, q, J ) 7.2 Hz), 2.72-2.81 (2H, m), 4.35-4.43 (1H, m), 7.03 (2H, d, J ) 8.8 Hz), 7.13 (2H, d, J ) 8.8 Hz), 7.44 (1H, t, J ) 8.0 Hz), 7.65 (1H, d, J ) 8.0 Hz), 7.74 (1H, t, J ) 8.0 Hz), 8.25 (1H, d, J ) 8.0 Hz); MS (ESI) m/z 364 (M + H)+. HRMS (M + H)+ calcd for C22H26N3O2, 364.2025; found, 364.2020.


2-Methyl-3-(4-[(1-methyl-4-piperidinyl)oxy]phenyl)-4(3H)quinazolinone (2a). Compound 2a was prepared from methyl iodide and 9b using the procedure described for 2b as a colorless solid (13% yield); HPLC purity (95.1%). 1H NMR (400 MHz, CDCl3) δ 1.91-1.99 (2H, br m), 2.06-2.18 (2H, br m), 2.27 (3H, s), 2.36-2.51 (2H, br m), 2.39 (3H, s), 2.74-2.82 (2H, br m), 4.40-4.46 (1H, br m), 7.05 (2H, d, J ) 8.8 Hz), 7.16 (2H, d, J ) 8.8 Hz), 7.47 (1H, t, J ) 7.8 Hz), 7.67 (1H, d, J ) 7.8 Hz), 7.77 (1H, td, J ) 7.8, 1.5 Hz), 8.27 (1H, dd, J ) 7.8, 1.5 Hz). MS (ESI) m/z 350 (M + H)+. HRMS (M + H)+ calcd for C21H24N3O2, 350.1869; found, 350.1865. 2-Methyl-3-(4-[(1-propyl-4-piperidinyl)oxy]phenyl)-4(3H)quinazolinone (2c). Compound 2c was prepared from n-propyl iodide and 9b using the procedure described for 2b as a colorless solid (48% yield); HPLC purity (95.6%). 1H NMR (400 MHz, CDCl3) δ; 0.93 (3H, t, J ) 7.3 Hz), 1.55-1.69 (2H, br m), 1.88-1.97 (2H, br m), 2.06-2.18 (2H, br m), 2.27 (3H, s), 2.31-2.49 (4H, br m), 2.76-2.85 (2H, br m), 4.37-4.46 (1H, br m), 7.04 (2H, d, J ) 8.8 Hz), 7.15 (2H, d, J ) 8.8 Hz), 7.47 (1H, td, J ) 7.8, 1.5 Hz), 7.67 (1H, d, J ) 7.8 Hz), 7.77 (1H, td, J ) 7.8, 1.5 Hz), 8.27 (1H, dd, J ) 7.8, 1.5 Hz). MS (ESI) m/z 378 (M + H)+. HRMS (M + H)+ calcd for C23H28N3O2, 378.2168; found, 378.2182. 3-(4-[(1-Butyl-4-piperidinyl)oxy]phenyl)-2-methyl-4(3H)-quinazolinone (2d). Compound 2d was prepared from n-butyl iodide and 9b using the procedure described for 2b as a colorless solid (43% yield); HPLC purity (99.8%). 1H NMR (400 MHz, CDCl3) δ 0.93 (3H, t, J ) 7.2 Hz), 1.28-1.39 (2H, m), 1.46-1.54 (2H, m), 1.83-1.93 (2H, m), 2.01-2.10 (2H, m), 2.26 (3H, s), 2.26-2.39 (4H, m), 2.72-2.81 (2H, m), 4.33-4.41 (1H, m), 7.03 (2H, d, J ) 8.8 Hz), 7.13 (2H, d, J ) 8.8 Hz), 7.44 (1H, t, J ) 8.0 Hz), 7.65 (1H, d, J ) 8.0 Hz), 7.74 (1H, t, J ) 8.0 Hz), 8.25 (1H, d, J ) 8.0 Hz); MS (ESI) m/z 392 (M + H)+. HRMS (M + H)+ calcd for C24H30N3O2, 392.2338; found, 392.2337. 3-(4-[(1-Isopropyl-4-piperidinyl)oxy]phenyl)-2-methyl-4(3H)quinazolinone (2e). Compound 2e was prepared from i-propyl iodide and 9b using the procedure described for 2b as a colorless solid (36% yield); HPLC purity (98.7%). 1H NMR (400 MHz, CDCl3) δ 1.07 (6H, d, J ) 6.4 Hz), 1.82-1.92 (2H, m), 2.02-2.11 (2H, m), 2.26 (3H, s), 2.37-2.46 (2H, m), 2.72-2.84 (3H, m), 4.31-4.40 (1H, m), 7.03 (2H, d, J ) 8.8 Hz), 7.13 (2H, d, J ) 8.8 Hz), 7.44 (1H, t, J ) 8.0 Hz), 7.65 (1H, d, J ) 8.0 Hz), 7.74 (1H, t, J ) 8.0 Hz), 8.25 (1H, d, J ) 8.0 Hz). MS (ESI) m/z 378 (M + H)+. HRMS (M + H)+ calcd for C23H28N3O2, 378.2182; found, 378.2186. Representative Procedure for the Preparation of 2f-l via Reductive Amination. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-2-methyl-4(3H)-quinazolinone (2f). To a stirred solution of 9b (370 mg, 1.10 mmol) and cyclobutanone (155 mg, 2.20 mmol) in methanol (10 mL) was added a solution of zinc chloride (409 mg, 3.00 mmol) and sodium cyanoborohydride (189 mg, 3.00 mmol) in methanol (6 mL) at room temperature, and the mixture was stirred at room temperature for 1 h. The resulting mixture was concentrated, and the residue was partitioned between ethyl acetate and water. The layers were separated, and the aqueous layer was extracted with ethyl acetate twice. The combined organic layers were washed with water, dried over sodium sulfate, and concentrated. The residue was purified by preparative HPLC to provide 2f as a colorless solid (165 mg, 39%); HPLC purity (99.6%). 1H NMR (400 MHz, CDCl3) δ 1.63-1.76 (2H, m), 1.84-1.95 (4H, m), 1.99-2.10 (4H, m), 2.14-2.23 (2H, m), 2.26 (3H, s), 2.59-2.67 (2H, m), 2.70-2.79 (1H, m), 4.33-4.41 (1H, m), 7.01 (2H, d, J ) 8.8 Hz), 7.44 (1H, t, J ) 8.0 Hz), 7.65 (1H, d, J ) 8.4 Hz), 7.74 (1H, t, J ) 7.6 Hz), 8.24 (1H, d, J ) 7.6 Hz). MS (ESI) m/z 390 (M + H)+. HRMS (M + H)+ calcd for C24H28N3O2, 390.2182; found, 390.2185. 3-(4-[(1-Cyclopentyl-4-piperidinyl)oxy]phenyl)-2-methyl-4(3H)quinazolinone (2g). Compound 2g was prepared from cyclopentanone and 9b using the procedure described for 2f as a colorless solid (42% yield); HPLC purity (95.4%). 1H NMR (400 MHz, CDCl3) δ 1.25-1.48 (2H, m), 1.52-1.61 (2H, m), 1.66-1.73 (2H, m), 1.83-1.93 (4H, m), 2.01-2.12 (2H, m), 2.25 (3H, s), 2.32-2.40 (2H, m), 2.49-2.58 (1H, m), 2.79-2.95 (2H, m), 4.33-4.41 (1H,


m), 7.03 (2H, d, J ) 8.8 Hz), 7.13 (2H, d, J ) 8.8 Hz), 7.44 (1H, t, J ) 7.2 Hz), 7.65 (1H, d, J ) 7.6 Hz), 7.74 (1H, t, J ) 7.2 Hz), 8.25 (1H, d, J ) 7.6 Hz). MS (ESI) m/z 418 (M + H)+. HRMS (M + H)+ calcd for C26H32N3O2, 418.2495; found, 418.2505. 3-(4-[(1-Cyclohexyl-4-piperidinyl)oxy]phenyl)-2-methyl-4(3H)quinazolinone (2h). Compound 2h was prepared from cyclohexanone and 9b using the procedure described for 2f as a colorless solid (48% yield); HPLC purity (99.5%). 1H NMR (400 MHz, CDCl3) δ 1.18-1.30 (6H, m), 1.78-1.90 (6H, m), 2.01-2.08 (2H, m), 2.26 (3H, s), 2.27-2.36 (1H, m), 2.42-2.51 (2H, m), 2.81-2.89 (2H, m), 4.30-4.38 (1H, m), 7.03 (2H, d, J ) 8.8 Hz), 7.13 (2H, d, J ) 8.8 Hz), 7.44 (1H, t, J ) 8.0 Hz), 7.65 (1H, d, J ) 8.0 Hz), 7.74 (1H, t, J ) 8.0 Hz), 8.25 (1H, d, J ) 8.0 Hz). MS (ESI) m/z 418 (M + H)+. HRMS (M + H)+ calcd for C26H32N3O2, 418.2495; found, 418.2505. 3-(4-[(1-Cyclobutyl-3-pyrrolidinyl)oxy]phenyl)-2-methyl-4(3H)quinazolinone (2i). Compound 2i was prepared from cyclobutanone and 9a using the procedure described for 2f as a beige solid (21% yield); HPLC purity (97.4%). 1H NMR (400 MHz, CDCl3) δ 1.68-1.78 (2H, m), 1.94-2.07 (5H, m), 2.25 (3H, s), 2.30-2.35 (1H, m), 2.46-2.52 (1H, m), 2.71-2.76 (2H, m), 2.86-2.91 (1H, m), 2.93-3.00 (1H, m), 4.83-4.87 (1H, m), 6.96 (2H, d, J ) 8.8 Hz), 7.12 (2H, d, J ) 8.8 Hz), 7.44 (1H, t, J ) 8.1 Hz), 7.64 (1H, d, J ) 8.1 Hz), 7.74 (1H, t, J ) 8.1 Hz), 8.24 (1H, d, J ) 8.1 Hz). MS (ESI) m/z 376 (M + H)+. HRMS (M + H)+ calcd for C23H26N3O2, 376.2025; found, 376.2019. 3-(4-[(1-Cyclopentyl-3-pyrrolidinyl)oxy]phenyl)-2-methyl-4(3H)quinazolinone (2j). Compound 2j was prepared from cyclopentanone and 9a using the procedure described for 2f as a colorless oil (18% yield); HPLC purity (97.9%). 1H NMR (400 MHz, CDCl3) δ 1.45-1.57 (4H, m), 1.66-1.88 (4H, m), 1.98-2.05 (1H, m), 2.25 (3H, s), 2.31-2.36 (1H, m), 2.46-2.52 (1H, m), 2.52-2.61 (1H, m), 2.79-2.84 (2H, m), 3.00-2.97 (1H, m), 4.82-4.87 (1H, m), 6.96 (2H, d, J ) 8.8 Hz), 7.12 (2H, d, J ) 8.8 Hz), 7.44 (1H, t, J ) 8.1 Hz), 7.64 (1H, d, J ) 8.1 Hz), 7.74 (1H, t, J ) 8.1 Hz), 8.24 (1H, d, J ) 8.1 Hz). MS (ESI) m/z 390 (M + H)+. HRMS (M + H)+ calcd for C24H28N3O2, 390.2182; found, 390.2185. 3-(4-[(1-Cyclobutyl-4-azepanyl)oxy]phenyl)-2-methyl-4(3H)quinazolinone (2k). Compound 2k was prepared from cyclobutanone and 9c using the procedure described for 2f as a pale-yellow oil (28% yield); HPLC purity (99.3%). 1H NMR (400 MHz, CDCl3) δ 1.57-2.18 (12H, m), 2.24 (3H, s), 2.39-2.50 (2H, m), 2.53-2.62 (2H, m), 2.85-2.95 (1H, m), 4.54-4.61 (1H, m), 6.96 (2H, d, J ) 8.8 Hz), 7.10 (2H, d, J ) 8.8 Hz), 7.43 (1H, t, J ) 8.0 Hz), 7.63 (1H, d, J ) 7.2 Hz), 7.72 (1H, t, J ) 7.2 Hz), 8.23 (1H, d, J ) 8.0 Hz). MS (ESI) m/z 404 (M + H)+. HRMS (M + H)+ calcd for C25H30N3O2, 404.2338; found, 404.2346. 3-(4-[(1-Cyclopentyl-4-azepanyl)oxy]phenyl)-2-methyl-4(3H)quinazolinone (2l). Compound 2l was prepared from cyclopentanone and 9c using the procedure described for 2f as a colorless oil (39% yield); HPLC purity (96.1%). 1H NMR (400 MHz, CDCl3) δ 1.55-2.22 (14H, br m), 2.26 (3H, s), 2.38-2.49 (1H, br m), 2.89-3.25 (4H, br m), 4.69-4.72 (1H, br m), 7.01 (2H, d, J ) 9.3 Hz), 7.16 (2H, d, J ) 8.8 Hz), 7.45-7.49 (1H, m), 7.67 (1H, d, J ) 7.8 Hz), 7.75-7.79 (1H, m), 8.27 (1H, dd, J ) 8.0, 1.2 Hz). MS (ESI) m/z 418 (M + H)+. HRMS (M + H)+ calcd for C26H32N3O2, 418.2495; found, 418.2491. 2-Methyl-3-[4-([3-(1-pyrrolidinyl)cyclopentyl]oxy)phenyl]-4(3H)quinazolinone (3a). A mixture of 12 (550 mg, 1.33 mmol), pyrrolidine (474 mg, 6.7 mmol), and potassium carbonate (277 mg, 2.0 mmol) in dry DMF (10 mL) was stirred at 80 °C for 14 h under a nitrogen atmosphere. The resulting mixture was poured into water and extracted with ethyl acetate three times. The combined organic extracts were washed with water, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography with 5% methanol in CHCl3 to give 3a as a colorless solid (143 mg, 28%); HPLC purity (99.2%). 1H NMR (400 MHz, CDCl3) δ 1.57-1.67 (1H, m), 1.79-1.82 (4H, m), 1.85-1.92 (2H, m), 2.03-2.08 (1H, m), 2.14-2.19 (1H, m), 2.22-2.30 (1H, m), 2.24 (3H, s), 2.53-2.56 (4H, br m), 2.76-2.83 (1H, m), 4.81-4.86 (1H, m), 6.96 (2H, d, J ) 8.8 Hz), 7.11 (2H,


d, J ) 8.8 Hz), 7.44 (1H, t, J ) 8.1 Hz), 7.66 (1H, d, J ) 8.1 Hz), 7.73 (1H, t, J ) 8.1 Hz), 8.23 (1H, d, J ) 8.1 Hz). MS (ESI) m/z 390 (M + H)+. HRMS (M + H)+ calcd for C24H28N3O2, 390.2182; found, 390.2166. 2-Methyl-3-[4-([3-(1-pyrrolidinyl)cyclopentyl]oxy)phenyl]-4(3H)quinazolinone (3b, Diastereomer of 3a). Compound 3b was prepared from 15 and pyrrolidine using the procedure described for 3a as a colorless solid (47% yield); HPLC purity (98.1%). 1H NMR (400 MHz, CDCl3) δ 1.77-1.96 (9H, m), 2.01-2.07 (1H, m), 2.26 (3H, s), 2.43-2.50 (1H, m), 2.56-2.61 (4H, br m), 4.80-4.74 (1H, m), 6.96-7.01 (2H, m), 7.13 (2H, d, J ) 8.8 Hz), 7.44-7.48 (1H, m), 7.67 (1H, d, J ) 8.3 Hz), 7.74-7.78 (1H, m), 8.27 (1H, dd, J ) 8.0, 1.2 Hz). MS (ESI) m/z 390 (M + H)+. HRMS (M + H)+ calcd for C24H28N3O2, 390.2182; found, 390.2179. cis-2-Methyl-3-(4-([4-(1-pyrrolidinyl)cyclohexyl]oxy)phenyl)4(3H)-quinazolinone (4a) and trans-2-Methyl-3-(4-([4-(1-pyrrolidinyl)cyclohexyl]oxy)phenyl)-4(3H)-quinazolinone (4b). Compounds 4a (cis-isomer) and 4b (trans-isomer) were synthesized from 18 and pyrrolidine using a similar procedure described for 2f. Purification by silica gel column chromatography with 20% f 50% ethyl acetate in hexanes followed by preparative HPLC afforded 4a as a colorless solid (260 mg, 46%) and 4b as a colorless solid (180 mg, 32%). The stereochemistry of each isomer was determined by NOE experiments. For cis-isomer (4a), HPLC purity (96.0%). 1 H NMR (400 MHz, CDCl3) δ 1.40-1.66 (4H, m), 1.77-1.81 (4H, m), 2.04-2.10 (3H, m), 2.12-2.20 (2H, m), 2.25 (3H, s), 2.60-2.63 (4H, br s), 4.19-4.26 (1H, m), 7.00 (2H, d, J ) 8.8 Hz), 7.11 (2H, d, J ) 8.8 Hz), 7.44 (1H, t, J ) 8.1 Hz), 7.65 (1H, d, J ) 8.1 Hz), 7.74 (1H, t, J ) 8.1 Hz), 8.25 (1H, d, J ) 8.1 Hz). MS (ESI) m/z 404 (M + H)+. HRMS (M + H)+ calcd for C25H30N3O2, 404.2338; found, 404.2351. For trans-isomer (4b), HPLC purity (98.2%). 1H NMR (400 MHz, CDCl3) δ 1.59-1.83 (10H, br m), 2.10-2.15 (3H, m), 2.26 (3H, s), 2.62 (4H, br s), 4.53-4.56 (1H, br m), 7.03 (2H, d, J ) 9.3 Hz), 7.13 (2H, d, J ) 8.8 Hz), 7.44-7.48 (1H, m), 7.67 (1H, d, J ) 7.8 Hz), 7.74-7.78 (1H, m), 8.27 (1H, dd, J ) 8.0, 1.2 Hz). MS (ESI) m/z 404 (M + H)+. HRMS (M + H)+ calcd for C25H30N3O2, 404.2338; found, 404.2343. Representative Procedure for the Preparation of 5a-s. 3-(4[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-2-methyl-5-(trifluoromethyl)-4(3H)-quinazolinone (5s). A mixture of 23 tosylate (20.0 g, 47.8 mmol), 2-methyl-5-(trifluoromethyl)-4H-3,1-benzoxazin4-one (24s; 10.95 g, 47.8 mmol) in acetic acid (110 mL) was stirred at room temperature for 30 h. The resulting mixture was concentrated, and the residue was partitioned between ethyl acetate and 2 N NaOH. The layers were separated, and the aqueous layer was extracted with ethyl acetate twice. The combined organic extracts were washed with 2 N NaOH and brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography with 0% f 2% methanol in CHCl3 followed by recrystallization from ethyl acetate to afford 5s as a colorless solid (13.7 g, 63%); HPLC purity (99.6%). 1H NMR (400 MHz, CDCl3) δ 1.63-1.77 (2H, m), 1.82-1.96 (4H, m), 1.98-2.09 (4H, m), 2.13-2.23 (2H, m), 2.26 (3H, s), 2.58-2.66 (2H, m), 2.70-2.79 (1H, m), 4.33-4.40 (1H, m), 7.01 (2H, d, J ) 8.8 Hz), 7.12 (2H, d, J ) 8.8 Hz), 7.77 (1H, d, J ) 8.0 Hz), 7.82-7.88 (2H, m). MS (ESI) m/z 458 (M + H)+. HRMS (M + H)+ calcd for C25H27N3O2F3, 458.2055; found, 458.2045. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-5-fluoro-2-methyl4(3H)-quinazolinone (5a). Compound 5a was prepared from 5-fluoro2-methyl-4H-3,1-benzoxazin-4-one (24a) and 23 tosylate using the procedure described for 5s as a colorless solid (50% yield); HPLC purity (99.2%). 1H NMR (400 MHz, CDCl3) δ 1.65-1.75 (2H, m), 1.83-1.93 (4H, m), 2.00-2.11 (4H, m), 2.13-2.24 (2H, m), 2.24 (3H, s), 2.60-2.68 (2H, m), 2.72-2.79 (1H, m), 4.36-4.41 (1H, m), 7.04 (2H, d, J ) 8.8 Hz), 7.08-7.14 (3H, m), 7.46 (1H, d, J ) 8.0 Hz), 7.65-7.71 (1H, m). MS (ESI) m/z 408 (M + H)+. HRMS (M + H)+ calcd for C24H27N3O2F, 408.2087; found, 408.2101.

Nagase et al.

3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-6-fluoro-2-methyl4(3H)-quinazolinone (5b). Compound 5bwas prepared from 6-fluoro2-methyl-4H-3,1-benzoxazin-4-one (24b) and 23 tosylate using the procedure described for 5s as a colorless solid (48% yield); HPLC purity (97.3%). 1H NMR (400 MHz, CDCl3) δ 1.63-1.75 (2H, m), 1.84-1.92 (4H, m), 2.01-2.12 (4H, m), 2.16-2.24 (2H, m), 2.25 (3H, s), 2.61-2.70 (2H, m), 2.72-2.81 (1H, m), 4.36-4.42 (1H, m), 7.04 (2H, d, J ) 8.8 Hz), 7.14 (2H, d, J ) 8.8 Hz), 7.45-7.51 (1H, m), 7.68 (1H, dd, J ) 4.8, 8.8 Hz), 7.89 (1H, dd, J ) 3.2, 8.0 Hz). MS (ESI) m/z 408 (M + H)+. HRMS (M + H)+ calcd for C24H27N3O2F, 408.2087; found, 408.2094. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-7-fluoro-2-methyl4(3H)-quinazolinone (5c). Compound 5c was prepared from 7-fluoro2-methyl-4H-3,1-benzoxazin-4-one (24c) and 23 tosylate using the procedure described for 5s as a colorless solid (72% yield); HPLC purity (99.8%). 1H NMR (400 MHz, CDCl3) δ 1.65-1.75 (2H, m), 1.82-1.96 (4H, m), 2.00-2.11 (4H, m), 2.14-2.24 (2H, m), 2.25 (3H, s), 2.60-2.68 (2H, m), 2.72-2.79 (1H, m), 4.36-4.41 (1H, m), 7.04 (2H, d, J ) 8.4 Hz), 7.12-7.20 (3H, m), 7.31 (1H, dd, J ) 2.0,9.6 Hz), 8.27 (1H, dd, J ) 6.0, 8.4 Hz). MS (ESI) m/z 408 (M + H)+. HRMS (M + H)+ calcd for C24H27N3O2F, 408.2087; found, 408.2090. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-8-fluoro-2-methyl4(3H)-quinazolinone (5d). Compound 5d was prepared from 8-fluoro-2-methyl-4H-3,1-benzoxazin-4-one (24d) and 23 tosylate using the procedure described for 5s as a colorless solid (60% yield); HPLC purity (99.0%). 1H NMR (400 MHz, CDCl3) δ 1.66-1.75 (2H, m), 1.84-1.95 (4H, m), 2.02-2.11 (4H, m), 2.17-2.28 (2H, m), 2.31 (3H, s), 2.61-2.68 (2H, m), 2.75-2.80 (1H, m), 4.37-4.43 (1H, m), 7.05 (2H, d, J ) 9.3 Hz), 7.14 (2H, d, J ) 9.3 Hz), 7.42-7.37 (1H, m), 7.47-7.52 (1H, m), 8.05 (1H, d, J ) 7.8 Hz). MS (ESI) m/z 408 (M + H)+. HRMS (M + H)+ calcd for C24H27N3O2F, 408.2087; found, 408.2106. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-6,7-difluoro-2methyl-4(3H)-quinazolinone (5e). Compound 5e was prepared from 6,7-difluoro-2-methyl-4H-3,1-benzoxazin-4-one (24e) and 23 tosylate using the procedure described for 5s as a colorless solid (67% yield); HPLC purity (99.6%). 1H NMR (400 MHz, CDCl3) δ 1.66-1.75 (2H, m), 1.83-1.95 (4H, m), 2.00-2.11 (4H, m), 2.16-2.24 (2H, m), 2.24 (3H, s), 2.60-2.68 (2H, m), 2.72-2.79 (1H, m), 4.35-4.42 (1H, m), 7.04 (2H, d, J ) 8.4 Hz), 7.13 (2H, d, J ) 8.4 Hz), 7.44 (1H, dd, J ) 6.8, 10.4 Hz), 8.01 (1H, dd, J ) 8.4, 9.6 Hz). MS (ESI) m/z 426 (M + H)+. HRMS (M + H)+ calcd for C24H26N3O2F2, 426.1993; found, 426.1998. 5-Chloro-3-(4-[(1-cyclobutyl-4-piperidinyl)oxy]phenyl)-2-methyl4(3H)-quinazolinone (5f). Compound 5f was prepared from 5-chloro2-methyl-4H-3,1-benzoxazin-4-one (24f) and 23 tosylate using the procedure described for 5s as a colorless solid (53% yield); HPLC purity (99.6%). 1H NMR (400 MHz, CDCl3) δ 1.63-1.77 (2H, m), 1.82-1.96 (4H, m), 1.99-2.09 (4H, m), 2.13-2.23 (2H, m), 2.23 (3H, s), 2.58-2.66 (2H, m), 2.69-2.79 (1H, m), 4.33-4.40 (1H, m), 7.00 (2H, d, J ) 8.8 Hz), 7.12 (2H, d, J ) 8.8 Hz), 7.43 (1H, dd, J ) 1.6, 7.2 Hz), 7.52-7.61 (2H, m). MS (ESI) m/z 424 (M + H)+. HRMS (M + H)+ calcd for C24H27N3O2Cl, 424.1792; found, 424.1785. 6-Chloro-3-(4-[(1-cyclobutyl-4-piperidinyl)oxy]phenyl)-2-methyl4(3H)-quinazolinone (5g). Compound 5g was prepared from 6-chloro2-methyl-4H-3,1-benzoxazin-4-one (24g) and 23 tosylate using the procedure described for 5s as a colorless solid (20% yield); HPLC purity (98.6%). 1H NMR (400 MHz, CDCl3) δ 1.66-1.79 (2H, m), 1.83-1.99 (4H, m), 2.00-2.12 (4H, m), 2.15-2.30 (5H, m), 2.59-2.71 (2H, m), 2.72-2.84 (1H, m), 4.40 (1H, br s), 7.04 (2H, d, J ) 8.8 Hz), 7.13 (2H, d, J ) 8.8 Hz), 7.61 (1H, d, J ) 8.8 Hz), 7.69 (1H, dd, J ) 8.8, 2.4 Hz), 8.22 (1H, d, J ) 2.4 Hz). MS (ESI) m/z 424 (M + H)+. HRMS (M + H)+ calcd for C24H27N3O2Cl, 424.1792; found, 424.1794. 7-Chloro-3-(4-[(1-cyclobutyl-4-piperidinyl)oxy]phenyl)-2-methyl4(3H)-quinazolinone (5h). Compound 5h was prepared from 7-chloro-2-methyl-4H-3,1-benzoxazin-4-one (24h) and 23 tosylate using the procedure described for 5s as a colorless solid (61% yield); HPLC purity (99.6%). 1H NMR (400 MHz, CDCl3) δ 1.65-1.74


(2H, m), 1.84-1.93 (4H, m), 2.01-2.08 (4H, m), 2.15-2.22 (2H, m), 2.25 (3H, s), 2.60-2.67 (2H, m), 2.71-2.79 (1H, m), 4.35-4.40 (1H, m), 7.04 (2H, d, J ) 9.3 Hz), 7.13 (2H, d, J ) 8.8 Hz), 7.41 (1H, dd, J ) 8.5, 2.2 Hz), 7.66 (1H, d, J ) 2.0 Hz), 8.19 (1H, d, J ) 8.8 Hz). MS (ESI) m/z 424 (M + H)+. HRMS (M + H)+ calcd for C24H27N3O2Cl, 424.1792; found, 424.1803. 8-Chloro-3-(4-[(1-cyclobutyl-4-piperidinyl)oxy]phenyl)-2-methyl4(3H)-quinazolinone (5i). Compound 5i was prepared from 8-chloro2-methyl-4H-3,1-benzoxazin-4-one (24i) and 23 tosylate using the procedure described for 5s as a colorless solid (69% yield); HPLC purity (99.0%). 1H NMR (400 MHz, CDCl3) δ 1.65-1.76 (2H, m), 1.85-1.93 (4H, m), 2.01-2.08 (4H, m), 2.15-2.22 (2H, m), 2.33 (3H, s), 2.60-2.67 (2H, m), 2.71-2.79 (1H, m), 4.41-4.36 (1H, m), 7.04 (2H, d, J ) 8.8 Hz), 7.13 (2H, d, J ) 8.8 Hz), 7.38 (1H, t, J ) 8.0 Hz), 7.84 (1H, dd, J ) 7.8, 1.5 Hz), 8.19 (1H, dd, J ) 8.3, 1.5 Hz). MS (ESI) m/z 424 (M + H)+. HRMS (M + H)+ calcd for C24H27N3O2Cl, 424.1792; found, 424.1786. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-5-methoxy-2-methyl4(3H)-quinazolinone (5j). Compound 5j was prepared from 5-methoxy-2-methyl-4H-3,1-benzoxazin-4-one (24j) and 23 tosylate using the procedure described for 5s as a beige solid (82% yield); HPLC purity (99.7%). 1H NMR (400 MHz, CDCl3) δ 1.63-1.77 (2H, m), 1.82-1.96 (4H, m), 1.98-2.09 (4H, m), 2.12-2.23 (2H, m), 2.21 (3H, s), 2.58-2.67 (2H, m), 2.69-2.79 (1H, m), 3.94 (3H, s), 4.33-4.40 (1H, m), 6.85 (1H, d, J ) 8.8 Hz), 6.99 (2H, d, J ) 8.8 Hz), 7.08 (2H, d, J ) 8.8 Hz), 7.21 (1H, d, J ) 8.0 Hz), 7.62 (1H, t, J ) 8.0 Hz). MS (ESI) m/z 420 (M + H)+. HRMS (M + H)+ calcd for C25H30N3O3, 420.2287; found, 420.2289. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-6-methoxy-2-methyl4(3H)-quinazolinone (5k). Compound 5k was prepared from 6-methoxy-2-methyl-4H-3,1-benzoxazin-4-one (24k) and 23 tosylate using the procedure described for 5s as a colorless solid (69% yield); HPLC purity (99.7%). 1H NMR (400 MHz, CDCl3) δ 1.64-1.74 (2H, m), 1.85-1.96 (4H, m), 2.01-2.13 (4H, m), 2.15-2.24 (2H, m), 2.23 (3H, s), 2.60-2.72 (2H, m), 2.72-2.81 (1H, m), 3.91 (3H, s), 4.35-4.42 (1H, brs), 7.04 (2H, d, J ) 6.8 Hz), 7.13 (2H, d, J ) 6.8 Hz), 7.36 (1H, dd, J ) 3.2, 9.2 Hz), 7.61 (1H, d, J ) 9.2 Hz), 7.63 (1H, d, J ) 3.2 Hz). MS (ESI) m/z 420 (M + H)+. HRMS (M + H)+ calcd for C25H30N3O3, 420.2287; found, 420.2302. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-7-methoxy-2-methyl4(3H)-quinazolinone (5l). Compound 5l was prepared from 7-methoxy-2-methyl-4H-3,1-benzoxazin-4-one (24l) and 23 tosylate using the procedure described for 5s as a pale-yellow solid (68% yield); HPLC purity (99.2%). 1H NMR (400 MHz, CDCl3) δ 1.65-1.76 (2H, m), 1.83-1.95 (4H, m), 2.00-2.09 (4H, m), 2.15-2.23 (2H, m), 2.24 (3H, s), 2.60-2.68 (2H, m), 2.71-2.79 (1H, m), 3.93 (3H, s), 4.35-4.41 (1H, m), 7.06-7.01 (4H, m), 7.14 (2H, d, J ) 9.3 Hz), 8.16 (1H, d, J ) 8.8 Hz). MS (ESI) m/z 420 (M + H)+. HRMS (M + H)+ calcd for C25H30N3O3, 420.2287; found, 420.2294. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-8-methoxy-2-methyl4(3H)-quinazolinone (5m). Compound 5m was prepared from 8-methoxy-2-methyl-4H-3,1-benzoxazin-4-one (24m) and 23 tosylate using the procedure described for 5s as a beige solid (87% yield); HPLC purity (98.8%). 1H NMR (400 MHz, CDCl3) δ 1.67-1.74 (2H, m), 1.84-1.93 (4H, m), 2.00-2.09 (4H, m), 2.15-2.22 (2H, m), 2.32 (3H, s), 2.61-2.66 (2H, m), 2.71-2.79 (1H, m), 4.04 (3H, s), 4.36-4.41 (1H, m), 7.03 (2H, d, J ) 8.8 Hz), 7.14 (2H, d, J ) 8.8 Hz), 7.22 (1H, dd, J ) 8.0, 1.2 Hz), 7.40 (1H, t, J ) 8.0 Hz), 7.85 (1H, dd, J ) 8.0, 1.2 Hz). MS (ESI) m/z 420 (M + H)+. HRMS (M + H)+ calcd for C25H30N3O3, 420.2287; found, 420.2295. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-2-methylpyrido[3,2d]pyrimidin-4(3H)-one (5n). Compound 5n was prepared from 2-methyl-4H-pyrido[3,2-d][1,3]oxazin-4-one (24n) and 23 tosylate using the procedure described for 5s as a pale-yellow solid (49% yield); HPLC purity (95.3%). 1H NMR (400 MHz, CDCl3) δ 1.67-1.74 (2H, m), 1.96-1.84 (4H, m), 2.03-2.10 (4H, m), 2.17-2.25 (2H, m), 2.27 (3H, s), 2.59-2.71 (2H, m), 2.73-2.79 (1H, m), 4.36-4.42 (1H, m), 7.05 (2H, td, J ) 2.0, 8.8 Hz), 7.17


(2H, td, J ) 2.0, 8.8 Hz), 7.67 (1H, dd, J ) 8.3, 4.4 Hz), 8.00 (1H, dd, J ) 8.3, 1.5 Hz), 8.84 (1H, dd, J ) 4.4, 1.5 Hz). MS (ESI) m/z 391 (M + H)+. HRMS (M + H)+ calcd for C23H27N4O2, 391.2134; found, 391.2144. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-2-methylpyrido[4,3d]pyrimidin-4(3H)-one (5o). Compound 5o was prepared from 2-methyl-4H-pyrido[4,3-d][1,3]oxazin-4-one (24o) and 23 tosylate using the procedure described for 5s as a pale-yellow solid (27% yield); HPLC purity (97.4%). 1H NMR (400 MHz, CDCl3) δ 1.63-1.77 (2H, m), 1.82-1.96 (4H, m), 1.99-2.11 (4H, m), 2.14-2.24 (2H, m), 2.29 (3H, s), 2.60-2.68 (2H, m), 2.70-2.80 (1H, m), 4.33-4.41 (1H, m), 7.03 (2H, d, J ) 8.8 Hz), 7.11 (2H, d, J ) 8.8 Hz), 7.47 (1H, d, J ) 5.2 Hz), 8.82 (1H, d, J ) 5.2 Hz), 9.45 (1H, s). MS (ESI) m/z 391 (M + H)+. HRMS (M + H)+ calcd for C23H27N4O2, 391.2134; found, 391.2122. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-2-methylpyrido[3,4d]pyrimidin-4(3H)-one (5p). Compound 5p was prepared from 2-methyl-4H-pyrido[3,4-d][1,3]oxazin-4-one (24p) and 23 tosylate using the procedure described for 5s as a colorless solid (11% yield); HPLC purity (98.1%). 1H NMR (400 MHz, CDCl3) δ 1.63-1.77 (2H, m), 1.82-1.95 (4H, m), 1.99-2.11 (4H, m), 2.15-2.23 (2H, m), 2.29 (3H, s), 2.60-2.69 (2H, m), 2.73-2.83 (1H, m), 4.36-4.43 (1H, m), 7.03 (2H, d, J ) 8.8 Hz), 7.12 (2H, d, J ) 8.8 Hz), 8.01 (1H, dd, J ) 0.8, 5.6 Hz), 8.65 (1H, d, J ) 5.2 Hz), 9.09 (1H, d, J ) 0.8 Hz). MS (ESI) m/z 391 (M + H)+. HRMS (M + H)+ calcd for C23H27N4O2, 391.2134; found, 391.2134. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-2-methylpyrido[2,3d]pyrimidin-4(3H)-one (5q). Compound 5q was prepared from 2-methyl-4H-pyrido[2,3-d][1,3]oxazin-4-one (24q) and 23 tosylate using the procedure described for 5s as a colorless solid (8% yield); HPLC purity (99.7%). 1H NMR (400 MHz, CDCl3) δ 1.63-1.77 (2H, m), 1.82-1.95 (4H, m), 1.99-2.11 (4H, m), 2.15-2.23 (2H, m), 2.34 (3H, s), 2.60-2.68 (2H, m), 2.70-2.80 (1H, m), 4.33-4.41 (1H, m), 7.03 (2H, d, J ) 8.8 Hz), 7.12 (2H, d, J ) 8.8 Hz), 7.39 (1H, dd, J ) 4.4, 8.0 Hz), 8.56 (1H, dd, J ) 2.0, 7.6 Hz), 8.96 (1H, dd, J ) 2.4, 4.8 Hz). MS (ESI) m/z 391 (M + H)+. HRMS (M + H)+ calcd for C23H27N4O2, 391.2134; found, 391.2122. 3-(4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl)-2,5-dimethyl-4(3H)quinazolinone (5r). Compound 5r was prepared from 2,5-dimethyl4H-3,1-benzoxazin-4-one (24r) and 23 tosylate using the procedure described for 5s as a colorless solid (70% yield); HPLC purity (99.9%). 1H NMR (400 MHz, CDCl3) δ 1.63-1.75 (2H, m), 1.82-1.96 (4H, m), 1.99-2.10 (4H, m), 2.13-2.22 (2H, m), 2.22 (3H, s), 2.58-2.67 (2H, m), 2.69-2.79 (1H, m), 2.81 (3H, s), 4.33-4.40 (1H, m), 7.02 (2H, d, J ) 8.8 Hz), 7.11 (2H, d, J ) 8.8 Hz), 7.19 (1H, d, J ) 8.0 Hz), 7.48 (1H, d, J ) 8.0 Hz), 7.57 (1H, t, J ) 8.0 Hz). MS (ESI) m/z 404 (M + H)+. HRMS (M + H)+ calcd for C25H30N3O2, 404.2338; found, 404.2343. 3-(4-[(1-tert-Butoxycarbonyl-4-piperidinyl)oxy]phenyl)-2-methyl4(3H)-quinazolinone (8b). To a stirred solution of 6 (1.0 g, 3.96 mmol), N-(tert-butoxycarbonyl)-4-piperidinol (7b; 956 mg, 4.75 mmol), and triphenylphosphine (1.56 g, 5.94 mmol) in THF (2 mL) was added diethyl azodicarboxylate (1.17 mL, 5.94 mmol) dropwise at 0 °C under a nitrogen atmosphere. The mixture was allowed to warm to room temperature and stirred for 48 h. The resulting mixture was concentrated, and the residual oil was taken up into diethyl ether. The resulting precipitates were removed by filtration, and the filtrate was concentrated. The residue was purified by silica gel column chromatography with 30% ethyl acetate in hexanes to give 8b as a light-brown solid (1.10 g, 64%). 1H NMR (400 MHz, CDCl3) δ 1.48 (9H, s), 1.76-1.84 (2H, br m), 1.92-1.99 (2H, br m), 2.26 (3H, s), 3.34 (2H, br m), 3.66-3.75 (2H, br m), 4.48-4.56 (1H, m), 7.03 (2H, d, J ) 9.2 Hz), 7.14 (2H, d, J ) 9.2 Hz), 7.51-7.55 (1H, m), 7.63 (1H, d, J ) 8.0 Hz), 7.72-7.77 (1H, m), 8.25 (1H, dd, J ) 8.0, 1.2 Hz). MS (ESI) m/z 436 (M + H)+. 3-(4-[(1-tert-Butoxycarbonyl-3-pyrrolidinyl)oxy]phenyl)-2-methyl-4(3H)-quinazolinone (8a). Compound 8a was prepared from compound 6 and N-(tert-butoxycarbonyl)-3-pyrrolidinol (7a) using the procedure described for 8b as a brown oil. 1H NMR (400 MHz, CDCl3) δ 1.49 (9H, s), 2.10-2.19 (1H, br m), 2.22-2.29 (1H, br m), 2.27 (3H, s), 3.50-3.70 (4H, br m), 4.93-4.96 (1H, br m),


7.03 (2H, d, J ) 7.3 Hz), 7.18 (2H, d, J ) 7.3 Hz), 7.45-7.49 (1H, m), 7.68 (1H, d, J ) 7.3 Hz), 7.77 (1H, t, J ) 7.3 Hz), 8.27 (1H, dd, J ) 8.0, 1.2 Hz). MS (ESI) m/z 422 (M + H)+. 3-(4-[(1-tert-Butoxycarbonyl-4-azepanyl)oxy]phenyl)-2-methyl4(3H)-quinazolinone (8c). Compound 8c was prepared from compound 6 and N-(tert-butoxycarbonyl)-4-azepanol (7c) using the procedure described for 8b as a brown oil. 1H NMR (400 MHz, CDCl3) δ 1.48 (9H, s), 1.65-1.70 (2H, br m), 1.93-2.02 (3H, m), 2.06-2.16 (1H, m), 2.25 (3H, s), 3.25-3.37 (1H, m), 3.40-3.64 (3H, m), 4.46-4.50 (1H, m), 6.98 (2H, d, J ) 8.8 Hz), 7.13 (2H, d, J ) 8.0 Hz), 7.44 (1H, t, J ) 8.4 Hz), 7.64 (1H, d, J ) 8.0 Hz), 7.71-7.75 (1H, m), 8.24 (1H, d, J ) 8.0 Hz). MS (ESI) m/z 450 (M + H)+. 2-Methyl-3-[4-(4-piperidinyloxy)phenyl]-4(3H)-quinazolinone (9b). To a stirred solution of 8b (1.10 g, 2.53 mmol) in CHCl3 (10 mL) was added trifluoroacetic acid (10 mL), and the mixture was stirred at room temperature for 30 min. The resulting mixture was concentrated, and the residue was partitioned between ethyl acetate and 2 N NaOH. The layers were separated, and the aqueous layer was extracted with ethyl acetate twice. The combined organic layers were washed with 2 N NaOH and brine, dried over sodium sulfate, and concentrated. The residue was vacuum-dried to give 9b as a slightly purple solid (0.83 g, 98%). 1H NMR (400 MHz, CDCl3) δ 1.67-1.81 (2H, m), 2.01-2.10 (2H, m), 2.26 (3H, s), 2.72-2.80 (2H, m), 3.13-3.20 (2H, m), 4.40-4.47 (1H, m), 7.03 (2H, d, J ) 8.8 Hz), 7.13 (2H, d, J ) 8.8 Hz), 7.44 (1H, t, J ) 8.0 Hz), 7.65 (1H, d, J ) 7.6 Hz), 7.74 (1H, t, J ) 7.6 Hz), 8.25 (1H, d, J ) 8.0 Hz). MS (ESI) m/z 336 (M + H)+. 2-Methyl-3-[4-(3-pyrrolidinyloxy)phenyl]-4(3H)-quinazolinone (9a). Compound 9a was prepared from 8a using the procedure described for 9b as a beige solid (34% over 2 steps from 6). 1H NMR (400 MHz, CDCl3) δ 2.01-2.20 (2H, m), 2.27 (3H, s), 2.94-3.01 (1H, br m), 3.06-3.11 (1H, br m), 3.19-3.28 (2H, m), 4.87-4.90 (1H, br m), 7.01 (2H, d, J ) 9.3 Hz), 7.16 (2H, d, J ) 9.3 Hz), 7.45-7.49 (1H, m), 7.67 (1H, d, J ) 7.8 Hz), 7.75-7.79 (1H, m), 8.27 (1H, dd, J ) 8.0, 1.2 Hz). MS (ESI) m/z 322 (M + H)+. 3-[4-(4-Azepanyloxy)phenyl]-2-methyl-4(3H)-quinazolinone (9c). Compound 9c was prepared from 8c using the procedure described for 9b as a brown oil (40% over 2 steps from 6). 1H NMR (400 MHz, CDCl3) δ 1.57-1.68 (1H, m), 1.83-2.22 (5H, m), 2.25 (3H, s), 2.84-3.06 (4H, m), 4.57-4.62 (1H, m), 6.99 (2H, d, J ) 8.8 Hz), 7.12 (2H, d, J ) 8.8 Hz), 7.43 (1H, t, J ) 8.0 Hz), 7.64 (1H, d, J ) 8.0 Hz), 7.74 (1H, t, J ) 8.0 Hz), 8.24 (1H, d, J ) 6.4 Hz). MS (ESI) m/z 350 (M + H)+. 3-(4-[(3-Hydroxycyclopentyl)oxy]phenyl)-2-methyl-4(3H)quinazolinone (11). To a stirred solution of 6 (1.0 g, 3.96 mmol), cyclopentane-1,3-diol (10; 810 mg, 7.93 mmol), and triphenylphosphine (1.56 g, 5.95 mmol) in THF (10 mL) was added diisopropyl azodicarboxylate (1.16 mL, 5.95 mmol) dropwise at 0 °C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 14 h. The resulting mixture was partitioned between water and ethyl acetate. The layers were separated, and the aqueous layer was extracted with ethyl acetate three times. The combined organic layers were washed with brine, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography with 50% f 100% ethyl acetate in hexanes to afford 11 (1.32 g, 99%) as a beige solid. 1H NMR (400 MHz, CDCl3) δ 1.91-2.20 (6H, m), 2.26 (3H, s), 4.38-4.42 (1H, br m), 4.85-4.89 (1H, br m), 7.03 (2H, d, J ) 8.8 Hz), 7.16 (2H, d, J ) 8.8 Hz), 7.46 (1H, td, J ) 7.4, 1.1 Hz), 7.67 (1H, d, J ) 7.8 Hz), 7.74-7.79 (1H, m), 8.27 (1H, dd, J ) 7.8, 1.0 Hz). MS (ESI) m/z 337 (M + H)+. 3-[4-((3-[(Methylsulfonyl)oxy]cyclopentyl)oxy)phenyl]-2-methyl4(3H)-quinazolinone (12). To a stirred solution of 11 (520 mg, 1.55 mmol) and triethylamine (0.33 mL, 2.32 mmol) in dichloromethane (10 mL) was added mesyl chloride (0.12 mL, 1.55 mmol) dropwise at 0 °C. After being stirred at 0 °C for 5 min, the mixture was allowed to warm to room temperature and stirred for an additional 10 min. The mixture was diluted with ethyl acetate and washed with water, dried over magnesium sulfate, and concentrated to give

Nagase et al.

12 as a pale-yellow solid (553 mg, 86%). 1H NMR (400 MHz, CDCl3) δ 2.05-2.30 (5H, m), 2.26 (3H, s), 2.44-2.51 (1H, m), 3.02 (3H, s), 4.80-4.83 (1H, m), 5.16-5.23 (1H, m), 6.99 (2H, d, J ) 9.2 Hz), 7.15 (2H, d, J ) 9.2 Hz), 7.42-7.46 (1H, m), 7.65 (1H, d, J ) 8.0 Hz), 7.74 (1H, t, J ) 8.4 Hz), 8.24 (1H, d, J ) 8.0 Hz). MS (ESI) m/z 415 (M + H)+. 3-[4-(2-Methyl-4-oxo-3(4H)-quinazolinyl)phenoxy]cyclopentyl benzoate (13). To a stirred solution of 11 (200 mg, 0.595 mmol), benzoic acid (109 mg, 0.892 mmol), and triphenylphosphine (234 mg, 0.892 mmol) in THF (2 mL) was added diisopropyl azodicarboxylate (173 µL, 0.892 mmol) dropwise at 0 °C under an nitrogen atmosphere. After being stirred at 0 °C for 10 min, the mixture was allowed to warm to room temperature and stirred for an additional 1 h. The resultant mixture was partitioned between water and ethyl acetate. The layers were separated, and the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography with 40% f 100% ethyl acetate in hexanes to give 13 (377 mg) as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 1.98-2.10 (2H, m), 2.23-2.46 (4H, m), 2.27 (3H, s), 4.96-5.03 (1H, br m), 5.59-5.63 (1H, m), 7.02 (2H, d, J ) 8.3 Hz), 7.16 (2H, d, J ) 8.8 Hz), 7.44-7.49 (3H, m), 7.57 (1H, t, J ) 7.3 Hz), 7.68 (1H, d, J ) 7.8 Hz), 7.75-7.79 (1H, m), 8.04 (2H, dd, J ) 8.5, 1.2 Hz), 8.28 (1H, dd, J ) 7.8, 1.0 Hz). MS (ESI) m/z 441 (M + H)+. 3-(4-[(3-Hydroxycyclopentyl)oxy]phenyl)-2-methyl-4(3H)quinazolinone (14, Diastereomer of 11). A mixture of 13 (227 mg, 0.36 mmol) and potassium carbonate (71.2 mg, 0.515 mmol) in methanol (5 mL) was stirred at room temperature for 8 h under a nitrogen atmosphere. The resulting mixture was partitioned between ethyl acetate and water. The layers were separated, and the aqueous layer was extracted with ethyl acetate twice. The combined organic layers were washed with 10% aqueous NaHCO3 and brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography with 60% f 100% ethyl acetate in hexanes to give 14 as a pale-yellow solid (80 mg, 66% over 2 steps from 11). 1H NMR (400 MHz, CDCl3) δ 1.68-1.75 (1H, m), 1.90-1.97 (1H, m), 2.07-2.21 (3H, m), 2.24-2.33 (1H, m), 2.26 (3H, s), 4.56-4.60 (1H, m), 4.93-4.98 (1H, m), 6.99 (2H, d, J ) 8.8 Hz), 7.14 (2H, d, J ) 8.8 Hz), 7.44-7.48 (1H, m), 7.67 (1H, d, J ) 7.3 Hz), 7.74-7.79 (1H, m), 8.27 (1H, dd, J ) 8.0, 1.2 Hz). MS (ESI) m/z 337 (M + H)+. 3-[4-((3-[(Methylsulfonyl)oxy]cyclopentyl)oxy)phenyl]-2-methyl4(3H)-quinazolinone (15, Diastereomer of 12). Compound 15 was prepared from 14 using the procedure described for 12 as a colorless solid (98% yield). 1H NMR (400 MHz, CDCl3) δ 2.01-2.13 (2H, m), 2.17-2.30 (2H, m), 2.26 (3H, s), 2.37-2.47 (2H, m), 3.04 (3H, s), 4.95-5.00 (1H, m), 5.32-5.37 (1H, m), 6.99 (2H, d, J ) 8.8 Hz), 7.16 (2H, d, J ) 9.3 Hz), 7.44-7.49 (1H, m), 7.68 (1H, d, J ) 7.8 Hz), 7.75-7.79 (1H, m), 8.27 (1H, dd, J ) 8.0, 1.2 Hz). MS (ESI) m/z 415 (M + H)+. 3-[4-(1,4-Dioxaspiro[4.5]dec-8-yloxy)phenyl]-2-methyl-4(3H)quinazolinone (17). Compound 17 was prepared from 6 and 4,4ethylenedioxycyclohexanol (16) using a similar procedure described for 8b as a pale-purple solid, which was used for the next reaction without further purification. 1H NMR (400 MHz, CDCl3) δ 1.62-1.69 (2H, m), 1.91-2.01 (6H, m), 2.27 (3H, s), 3.96-4.01 (4H, m), 4.45-4.49 (1H, m), 7.05 (2H, d, J ) 9.3 Hz), 7.15 (2H, d, J ) 9.3 Hz), 7.46 (1H, t, J ) 7.6 Hz), 7.67 (1H, d, J ) 7.8 Hz), 7.74-7.79 (1H, m), 8.27 (1H, dd, J ) 8.0, 1.2 Hz); MS (ESI) m/z 393 (M + H)+. 2-Methyl-3-(4-[(4-oxocyclohexyl)oxy]phenyl)-4(3H)-quinazolinone (18). To a solution of 17 (1.59 g) in THF (5 mL) was added 10% hydrochloric acid (10 mL), and the mixture was stirred at room temperature for 3 h. After being neutralized with 2 N NaOH, the mixture was extracted with ethyl acetate three times. The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography with 5% methanol in CHCl3 to give 18 as a light-orange solid (511 mg, 49% yield for 2 steps). 1H NMR


(400 MHz, CDCl3) δ 2.08-2.16 (2H, m), 2.27 (3H, s), 2.30-2.40 (4H, m), 2.67-2.76 (2H, m), 4.73-4.78 (1H, m), 7.09 (2H, d, J ) 8.4 Hz), 7.18 (2H, d, J ) 8.8 Hz), 7.45 (1H, t, J ) 8.0 Hz), 7.65 (1H, d, J ) 7.2 Hz), 7.73-7.77 (1H, m), 8.25 (1H, dd, J ) 8.0, 1.2 Hz). MS (ESI) m/z 349 (M + H)+. 1-tert-Butoxycarbonyl-4-(4-nitrophenoxy)piperidine (20). To a stirred solution of N-(tert-butoxycarbonyl)-4-piperidinol (7b; 10.0 g, 49.7 mmol) in DMF (50 mL) was added sodium hydride (60% dispersion in mineral oil, 3.0 g, 75 mmol) portionwise at 0 °C under a nitrogen atmosphere. After being stirred at 0 °C for 15 min, the mixture was allowed to warm to room temperature and stirred for an additional 30 min. To the mixture was added a solution of 4-fluoronitrobenzene (7.71 g, 55 mmol) in DMF (10 mL) dropwise at 0 °C. The mixture was allowed to warm to room temperature and stirred for 15 h. After being quenched by the addition of water (15 mL), the mixture was concentrated. The residue was partitioned between ethyl acetate (120 mL) and water (120 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate twice. The combined organic layers were washed with water twice, dried over sodium sulfate, and concentrated. The residual solid was suspended in a mixture of diisopropylether/hexanes (v/v ) 1/4), and the precipitates were collected by filtration and vacuumdried to provide 20 as a pale-yellow solid (12.4 g, 77%). 1H NMR (400 MHz, CDCl3) δ 1.48 (9H, s), 1.75-1.82 (2H, m), 1.93-1.99 (2H, m), 3.36-3.42 (2H, m), 3.66-3.73 (2H, m), 4.58-4.62 (2H, m), 6.96 (2H, d, J ) 8.0 Hz), 8.20 (2H, d, J ) 8.0 Hz). MS (ESI) m/z 323 (M + H)+. 4-(4-Nitrophenoxy)piperidine (21). Compound 20 (10.9 g, 33.8 mmol) was dissolved in 30 mL of trifluoroacetic acid, and the mixture was stirred at room temperature for 1 h. The resulting mixture was concentrated, and the residue was partitioned between ethyl acetate (150 mL) and 2 N NaOH (100 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate twice. The combined organic layers were washed with 1 N NaOH and brine, dried over sodium sulfate, and concentrated to give 21 as a brown oil (7.5 g, 99%). 1H NMR (400 MHz, CDCl3) δ 1.70-1.79 (2H, m), 2.03-2.09 (2H, m), 2.77-2.83 (2H, m), 3.14-3.20 (2H, m), 4.49-4.55 (1H, m), 6.95 (2H, d, J ) 8.0 Hz), 8.19 (2H, d, J ) 8.0 Hz). MS (ESI) m/z 223 (M + H)+. 1-Cyclobutyl-4-(4-nitrophenoxy)piperidine (22). To a mixture of 21 (7.5 g, 33.8 mmol), zinc chloride (4.61 g, 33.8 mmol), and cyclobutanone (3.55 g, 50.7 mmol) in methanol (100 mL) was added sodium cyanoborohydride (3.19 g, 50.7 mmol) portionwise at room temperature, and the mixture was stirred at room temperature for 5 h. The resulting mixture was concentrated, and the residue was partitioned between ethyl acetate and 2 N NaOH. The layers were separated, and the aqueous layer was extracted with ethyl acetate twice, and the combined organic layers were washed with 1 N NaOH and brine, dried over sodium sulfate, and concentrated. The resulting solid was suspended in a mixture of diisopropylether/hexanes (v/v ) 1/4), and the precipitates were collected by filtration and vacuum-dried to give 22 (7.5 g, 80%) as a pale-yellow solid. 1H NMR (400 MHz, CDCl3) δ 1.75-1.86 (1H, m), 1.87-1.94 (1H, m), 2.07-2.17 (2H, br m), 2.22-2.35 (6H, br m), 2.92-3.02 (4H, br m), 3.28-3.34 (1H, m), 4.76 (1H, br s), 6.99 (2H, d, J ) 9.2 Hz), 8.18 (2H, d, J ) 9.2 Hz). MS (ESI) m/z 277 (M + H)+. 4-[(1-Cyclobutyl-4-piperidinyl)oxy]aniline p-Toluenesulfonate (23 Tosylate). Compound 22 (7.5 g, 27 mmol) was hydrogenated over 3.0 g of 10% Pd/C in methanol (100 mL) under a hydrogen atmosphere (1 atm) at room temperature for 13 h. The mixture was filtered through a pad of celite, and the filtrate was concentrated. The residue was vacuum-dried to give the aniline 23 as a palebrown oil (6.5 g, 97%). To a stirred solution of 23 (6.5 g, 26.4 mmol) in ethyl acetate (100 mL) was added a solution of p-toluenesulfonic acid monohydrate (26.4 mmol) in EtOH (20 mL) at room temperature. The mixture was heated to reflux for 10 min to dissolve the precipitates. After being cooled to room temperature, the resulting precipitates were collected and vacuum-dried to provide 23 tosylate as a beige solid (10.3 g, 93%). 1H NMR (400 MHz, CDCl3/CD3OD ) 4/1) δ 1.68-1.79 (1H, m), 1.81-1.90 (1H,


m), 2.09-2.15 (2H, m), 2.19-2.27 (2H, m), 2.34-2.43 (5H, m), 2.52-2.61 (2H, m), 2.86-2.93 (2H, m), 3.34-3.44 (3H, m), 4.52 (1H, brs), 6.70-6.75 (4H, m), 7.21 (2H, d, J ) 7.8 Hz), 7.79 (2H, d, J ) 7.8 Hz). MS (ESI) m/z 247 (M + H)+. Cloning and Expression of the Histamine H3 Receptor. Human histamine H3 receptor (GenBank accession no. AB045369) was cloned as previously described.32 The DNAs were inserted into the mammalian expression vector pCAGGS33 (pCAGGS-hH3), and cells, stably expressing the human histamine H3 receptors, were generated by transfecting pCAGGS-hH3 into CHO-K1 cells. HEK293/CRE-β-lactamase cells expressing rat histamine H3 receptor were prepared as previously described.34 Ligand Binding Assay. Membrane preparations from the cells expressing histamine H3 receptors and [3H] N-R-methylhistamine binding studies were performed as previously described.34 [35S]GTPγS Functional Binding Assay. [35S]GTPγS functional binding assays were carried out with a minor modification to the method described previously.34 In brief, membranes were incubated with 200 pM [35S]GTPγS in 50 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, and 10 µM GDP (pH 7.4) containing 1.0 mg of wheat germ agglutinin-coated SPA beads for 3 h at 25 °C in the presence or absence of various concentrations of compounds. Membranebound radioactivity was detected by scintillation proximetry with a TopCount microplate scintillation counter (Packard, Meriden, CT). Brain Histamine Release Assay. Brain tele-methylhistamine levels were measured as previously described.29 In brief, 2 h after treatment with orally administered vehicle (0.5% methylcellulose) or compounds 5a and 5s (1, 3, and 10 mg /kg), male SD rats (Charles River, Tokyo, Japan) were decapitated and the brains were homogenized. After the solid phase extraction, tele-methylhistamine was analyzed by high performance liquid chromatography with fluorometry. Receptor Occupancy by Ex Vivo Autoradiography. Male SD rats received vehicle or compounds 5a and 5s (1 mg /kg) by oral gavage. Animals were decapitated 2 h following oral dosing. In other studies, female mdr1a (+/+) and mdr1a (-/-) CF-1 mice (Charles River, Tokyo, Japan) were administered vehicle or compound 5a (0.1, 0.3, and 1 mg /kg) or 5s (0.3, 1, and 3 mg/kg) by oral gavage and decapitated 2 h following oral dosing. Terminal blood samples were collected and whole brains were rapidly removed in all studies. Whole brain tissue was dissected from half of each brain (for ex vivo autoradiography), and the other half of the brain was kept for pharmacokinetic analysis of brain concentrations of 5a and 5s. All dissected tissue samples were frozen in dry ice-cold isopentane and stored at -80 °C until use. Serial coronal sections, 20 µm in thickness, of the anterior striatal (0.2-1.0 mm anterior to Bregma) regions were cut by cryostat microtome and stored at -80 °C. Striatal sections were incubated with 400 pM of 3-([1,1,1-3H]methyl)-2-(4-([3-(1-pyrrolidinyl)propyl]oxy)phenyl)4(3H)-quinazolinone (a selective histamine H3 receptor inverse agonist radioligand) in sodium phosphate buffer for 10 min at room temperature. Nonspecific binding was defined by the presence of R-R-methylhistamine (10 µM). Following incubation, the striatum sections were washed, air-dried, and exposed to beta-Imager (Biospace, Paris, France) for 6 h. Autoradiographic images were quantified as photostimulated luminescence (cpm) per mm2 by betaImager. Blood samples were centrifuged to separate the plasma, and the brain samples were homogenized by ultrasonification with 4 volumes of water. The plasma and brain homogenate samples were deproteinized with ethanol containing an internal standard. Compounds 5a and 5s and the internal standard were detected by LC-MS/MS in a positive ionization mode using the electrospray ionization probe, and their precursor to product ion combinations were monitored in Multiple Reaction Monitoring mode. Pharmacokinetics. Pharmacokinetic characterizations were conducted in male SD rats, male Beagle dogs, and male rhesus monkeys following single oral and single intravenous administration. In the three species, single doses of 5a, 5r, and 5s were administered either intravenously in a vehicle of PEG400/EtOH/H2O ) 50/10/40 or orally by gavage in a vehicle of 0.5% methylcellurose aqueous suspension. Doses of 1 (iv) and 3 (po) mg/kg for rats and 0.3 (iv)


and 1 (po) mg/kg for dogs and monkeys were used. Blood samples for the determination of drug plasma concentrations were obtained at multiple time points up to 24 h after administration. Blood samples were centrifuged to separate the plasma, and the plasma samples were deproteinized with ethanol containing an internal standard. Compounds 5a, 5r, and 5s and the internal standard were detected by LC-MS/MS in a positive ionization mode using the electrospray ionization probe, and their precursor to product ion combinations were monitored in Multiple Reaction Monitoring mode.

Acknowledgment. We thank Hirokazu Ohsawa for collecting the high-resolution mass spectral data, Hiroaki Suwa for HPLC purity analyses, and Dr. Takahiro Fukuroda for cardiovascular studies in anesthetized dogs. We also thank Drs. Nobuyoshi Yasuda, Robert J. DeVita, and Peter T. Meinke (Merck Research Laboratories, Rahway, NJ) for the editing of this manuscript. Supporting Information Available: Preparation of benzoxazinones 24e and 24g-i and procedures for safety-related studies, HPLC retention times and purity for the target compounds, and HPLC traces for 2f, 5a, 5r, and 5s. This material is available free of charge via the Internet at http://pubs.acs.org.

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Synthesis and Evaluation of Structurally Constrained Quinazolinone

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